DESCRIPTION: This project addresses the mechanism and regulation of vitamin B12 uptake in Escherichia coli. Both vitamin B12 and the iron siderophore complexes are normally present at low concentrations and are too large for entry through the outer membrane porin channels of most gram negative bacteria. Their entry requires the unusual operation of high affinity, energy dependent transport proteins in the outer membrane. The TonB/ExbB/ExbD complex appears to couple metabolic energy to the cobalamin transport protein BtuB. Transport of cobalamins from the periplasm into the cytoplasm operates by an ATP-dependent process that requires the BtuC and BtuD proteins and resembles the class of periplasmic permeases. This project continues the molecular genetic and biochemical analysis of the BtuB protein, with the aim of identifying the segments of the protein that determine substrate specificity, energy coupling and interaction with TonB protein, regulation by a phage BF23 encoded lipoprotein, and outer membrane localization. Specific objectives are to employ various mutagenesis procedures to investigate the role of specific residues that are conserved among the family of TonB dependent outer membrane transporters or that flank existing mutations that affect function. Intragenic and extragenic suppressor mutations that compensate for the transport defect of selected btuB mutations will be isolated and characterized to obtain insight into interactions among component proteins of the transport process. A variety of topological reporters will be employed to test predictions of the transmembrane orientation of the BtuB protein in the outer membrane. The BtuB protein will be purified and the conditions for its reconstitution in active form into proteoliposomes will be systematically determined. The ligand binding properties and the possible existence of a transmembrane channel in the BtuB protein which might be subject to gating by cobalamins or other effectors will be pursued. The repression of BtuB synthesis by vitamin B12 occurs at post transcriptional levels. This unusual regulatory process requires the presence of sequences within a long leader region and others within the coding sequence itself. Specific models of coupled attenuation and translational control mediated through changes in RNA structure will be tested by construction of mutations and compensating changes, by probing of the secondary structure of the leader RNA, and by testing the action on gene expression of isolated segments of the regulatory region.